Investigating CHD3-associated craniofacial syndromes

Context and challenge the proposal addresses: Craniofacial malformations, resulting from abnormal embryonic development, account for over one-third of all congenital birth defects. Cranial neural crest cells (CNCCs) are a developmental cell type essential during craniofacial development, as they differentiate into various structures in the skull, including craniofacial bones, cartilage and connective tissues. CNCC specification and differentiation into derivatives is regulated by fine-tuned gene expression, which is influenced by the accessibility of chromatin. Chromatin remodellers, like the Nucleosome Remodelling and Deacetylase Complex (NuRD) can modify chromatin accessibility. Mutations in CHD3 are the only known cause of Snijders Blok Campeau Syndrome, a congenital disorder characterized by distinctive craniofacial abnormalities. Moreover, CHD3 is one of the most frequently mutated genes in cleft lip and palate patients. Currently the cellular and molecular mechanisms by which CHD3 alterations lead to craniofacial defects are unknown. We are in possession of unique resources to address this knowledge gap, including Snijders Blok Campeau patient-derived iPSCs with CHD3 mutations, CRISPR iPSC lines with CHD3 heterozygous and homozygous knock out, a CHD3-dTAG iPSC line, two different mouse models with Snijders Blok Campeau patient-specific mutations, and CHD3fl/fl mouse models. Our preliminary data indicate that CHD3 regulates response to BMP signalling pathway. The latter is one of the key signalling pathways involved in both CNCC specification and in craniofacial osteoblast formation. Therefore, we will use these tools to investigate the role of CHD3 in CNCC specification and in CNCC-to-osteoblast differentiation, and more broadly to elucidate the molecular mechanisms underlying the craniofacial abnormalities typical of the CHD3-associated syndromes. Hypothesis: We hypothesise that CHD3 depletion impairs the ability of the cells to respond to BMP signalling. As a result, the differentiating cells undergo mesodermal fate upon exposure to Wnt signalling, and more specifically because of a Wnt/BMP imbalance. Aims: Define the genetic requirements for CHD3 in CNCC specification and differentiation. Model the functional consequence of CHD3 patient variants on development of the CNCC lineage. To investigate the regulation of BMP responses by CHD3 during craniofacial skeletogenesis Potential applications and benefits: The findings will advance our understanding of CHD3's function in craniofacial development and its broader implications in congenital disorders involving chromatin remodelling mechanisms. Understanding tissue-specific dynamics of the CHD3-NuRD complex will provide insight into how major signalling pathways essential for development integrate with chromatin remodellers, and thus, shed light on other diseases associated with dysregulation of the same genes and pathways. The proposal will have a major impact on our understanding of craniofacial development and has the potential to elucidate target genes potentially amenable to pharmacological treatment applicable to a wide range of diseases. Importantly, this proposal has the potential to generate insights into the molecular mechanisms underlying the syndrome, hence benefiting the patients and their families.